Evaluation of the toxicity of Caralluma europaea (C.E) extracts and their effects on apoptosis and chemoresistance in pancreatic cancer cells

Figures & data

Figure 1. Aerial parts of C. europaea: ((a) Stem; (b) Flower).

Table 1. Receptor grid coordinates of human Smo receptors (PDB ID).

	Human Smo receptors (PDB ID)	5L7I	4QIM	4QIN	4O9R	4N4W
Receptor grid coordinates	X	5.433	13.959	−21.422	49.012	−13.632
	Y	56.226	−12.636	61.863	8.014	−21.057
	Z	−38.917	−22.477	−1.461	48.944	−9.046

Figure 2. LC–MS/MS results based on AUC (Area Under Curve) of Caralluma europaea hydroethanolic extract phenolic compounds through direct infusion.

Table 2. Principal phenolic compounds revealed in C. europaea hydroethanolic extract through direct infusion.

Molecule	m/z	AUC
Quercetin	301.0000	1017541
Quercetin-3-O-hexose deoxyhexose	609.1000	1191028
Hesperetin	301.3000	2842636
Trimethoxyflavone	312.0000	215074
Isorhamnetin- 3-O-rutinoside	623.1000	1159286
Isorhamnetin-7-O- pentose	447.1000	4458497
Luteolin	284.9000	40808265
Luteolin 7-O-glucoside	447.1000	4345376
Kaempferol	285.0000	62076400
Kaempferol-3-O-glucose	609.1000	1262846
Kaempferol-3-O-glucuronic acid	461.1000	1041713
Kaempferol-3-O-pentose	417.1000	1771005
Kaempferol-3-O-hexose deoxyhexose	593.1000	14508482
Tyrosol	153.4000	521297
Gallic acid	168.9000	79633
Syringic acid	197.0000	1838400
Caffeic acid	179.0000	2517767
Sinapic acid	223.0000	2324483
Ferulic acid	193.0000	2405840
Trans ferulic acid	193.0000	3657099

Table 3. Effect of C. europaea hydroethanolic extract on weekly body weight gain of rat groups (n = 5). Values expressed as mean ± SEM.

	Acute toxicity
Parameters	Control	100 mg/kg	300 mg/kg	2000 mg/kg	5000 mg/kg
Initial weight (g)	188.4 ± 0.342	190.5 ± 0.646	193.8 ± 1.315	141.3 ± 1.031	199.7 ± 3.180
One week (g)	199.6 ± 0.963	201.8 ± 0.854	209.8 ± 1.250	147.0 ± 1.225	212.7 ± 2.728
Two weeks (g)	210.6 ± 0.457	212 ± 0.913	217.3 ± 1.109	163.5±0.646	222.0 ± 3.215
Body weight gain (g)	22.23 ± 0.202	21.50 ± 0.289^ns	23.25 ± 1.109^ns	22.25 ± 0.479^ns	23.33 ± 0.667^ns

ns: No significant

Table 4. Effect of C. europaea extracts on weekly body weight gain of rat groups (n = 5). Values expressed as mean ± SEM.

	Subacute toxicity
Parameters	Control	Total extract (100 mg/kg)	Polyphenol (50 mg/kg)	Flavonoid (15 mg/kg)	Saponins (10 mg/kg)
Initial weight (g)	150.3 ± 0.882	148 ± 3.512	156.3 ± 1.667	122.3 ± 0.854	124.0 ± 0.577
One week (g)	157.7 ± 1.764	157.7 ± 3.712	160.0 ± 4.000	127.3 ± 5.793	131.0 ± 1.732
Two weeks (g)	167.0 ± 1.155	170.3 ± 2.906	175.0 ± 6.429	138.0 ± 5.212	138.0 ± 1
Three weeks (g)	173.7 ± 0.882	178.5 ± 1.500	180.3 ± 4.842	146.8 ± 5.170	145.3 ± 2.603
Final weight (g)	183.7 ± 1.856	182.7 ± 3.844	189.0 ± 5.033	155.0 ± 5.050	150.7 ± 2.333
Body weight gain (g)	33.33 ± 2.728	34.67 ± 0.333^ns	32.67 ± 4.256^ns	32.75 ± 4.535^ns	26.67 ± 1.764^ns

Table 5. The relative organ weight (ROW) results of rats treated orally with C. europaea hydroethanolic extract. Values are expressed as mean ± SEM (n = 5).

	Acute toxicity
Organ	Control	100 mg/kg	300 mg/kg	2000 mg/kg	5000 mg/kg
Kidney	8.170 ± 0.222	7.249 ± 0.132^ns	7.307 ± 0.195^ns	7.513 ± 0.244^ns	7.460 ± 0.352^ns
Liver	41.13 ± 0.633	40.00 ± 2.309^ns	42.33 ± 2.028^ns	38.30 ± 1.457^ns	36.00 ± 2.405^ns
Spleen	2.960 ± 0.047	2.808 ± 0.483^ns	2.382 ± 0.111^ns	2.194 ± 0.093^ns	3.103 ± 0.511^ns

Table 6. The relative organ weight (ROW) results of rats treated orally with C. europaea extracts. Values are expressed as mean ± SEM (n = 5).

	Subacute toxicity
Organ	Control	Total extract (100 mg/kg)	Polyphenol (50 mg/kg)	Flavonoid (15 mg/kg)	Saponins (10 mg/kg)
Kidney	7.53 ± 0.233	6.809 ± 0.392^ns	6.479 ± 0.466^ns	6.366 ± 0.184^ns	7.705 ± 0.503^ns
Liver	44.13 ± 1.198	37.89 ± 3.161^ns	41.51 ± 4.445^ns	48.34 ± 1.403^ns	47.14 ± 4.036^ns
Spleen	3.13 ± 0.297	2.01 ± 0.073^ns	2.552 ± 0.503^ns	2.425 ± 0.142^ns	3.026 ± 0.561^ns

Table 7. Effects of C. europaea hydroethanolic extract on the liver (aspartate transaminase (AST), alanine aminotransferase (ALT)), and the kidney (creatinine (CREA) and urea) quantification during the acute toxicity test. (*) indicates a significant difference at p < 0.05 from the control group using one-way ANOVA followed by Dunnett test. Values are expressed as mean ± SEM (n = 5).

	Acute toxicity
Parameters	Control	100 mg/kg	300 mg/kg	2000 mg/kg	5000 mg/kg
AST (U/l)	163.6 ± 3.190	177.7 ± 4.978^ns	179±5.196 ^ns	171.7±4.410^ns	199.0 ± 14.54*
ALT (U/l)	44.60 ± 0.404	34.00 ± 4.041^ns	38.33±6.766^ns	38±3.055^ns	40.60 ± 7.200^ns
CREA (mg/l)	3.667 ± 0.433^ns	4.667 ± 0.333^ns	4.657±0.328^ns	4.223±0.223^ns	3.567±0.273^ns
Urea (g/l)	0.309 ± 0.008	0,253 ± 0.089^ns	0.233±0.018^ns	0.265±0.005^ns	0.273±0.062^ns

Table 8. Effects of hydroethanolic, polyphenols, flavonoids and saponins extracts on the liver (aspartate transaminase (AST), alanine aminotransferase (ALT)), and the kidney (creatinine (CREA) and urea) quantification during the subacute toxicity test.

	Subacute toxicity
Parameters	Control	hydroethanolic extract (100 mg/kg)	Polyphenol (50 mg/kg)	Flavonoids (15 mg/kg)	Saponins (10 mg/kg)
AST (U/l)	151.2 ± 9.040	158.7 ± 11.98^ns	149.7 ± 5.755^ns	158.0 ± 2.000^ns	189.0 ± 6.658*
ALT (U/l)	50.30 ± 2.601	49.00 ± 7.670^ns	51.67 ± 6.741^ns	50.67 ± 11.89^ns	55.00 ± 18.68*
CREA (mg/l)	3.843 ± 0.289	4.450 ± 0,450^ns	3.250 ± 0.150^ns	3.233 ± 0.285^ns	3.855 ± 0.355^ns
Urea (g/l)	0.291 ± 0.020	0.390 ± 0.050^ns	0.359 ± 0.069^ns	0.355 ± 0.015^ns	0.357 ± 0.033^ns

(*) indicates a significant difference at p < 0.05 from the control group using one-way ANOVA followed by Dunnett test. Values are expressed as mean ± SEM (n = 5).

Figure 3. Histopathological photomicrographs of kidney and liver slices stained with hematoxylin-eosin (×40) after oral administration of C. europaea hydroethanolic, polyphenols, flavonoids and saponins extracts in rats on day 28 of the subacute toxicity. (A–E) Kidney section of groups treated with NaCl, hydroethanolic, polyphenol, flavonoid and saponin extracts, respectively. (F–J) Liver section of groups treated with NaCl, hydroethanolic, polyphenol, flavonoid and saponin extracts, respectively. A, Arteriole; G, Glomerulus; CPT, Peritubular capillaries; VC, Central vein; VP, Portal vein; CB, Bile duct; HA, Hepatic artery; H, Hepatocyte; N, Neutrophil; L, Leukocyte.

Figure 4. Bx-PC3 (A) and MIA PaCa-2 (B) cells were cultured for 72 h in the presence of C. europaea extracts with different doses of total extract, polyphenols, flavonoids and saponins. Cell survival was measured by MTT assay. Values are expressed as mean ± SEM (n = 3). *p < 0.05 compared to the control.

Figure 5. Photomicrographs of Bx-PC3 (A) and MIA PaCa-2 (B) cells treated with various doses of C. europaea extracts. Images 10x.

Figure 6. Bx-PC3 (A) and MIA PaCa-2 (B) cells were incubated with C. europaea extracts alone, and in combination with gemcitabine. Values are expressed as mean ± SEM (n = 3). *p < 0.05 compared to the control. 

Figure 7. Bx-PC3 (A) and MIA PaCa-2 (B) were cultured for 72 h with C. europaea extracts. Protein levels of Nanog, Sox-2 and CD133 were determined by western blotting. GAPDH was used to demonstrate equal loading, and for purpose of quantifying the ratio of the band intensities.

Figure 8. Internucleosomal DNA fragmentation. (A) MIA PaCa-2 cells; (B) MIA PaCa-2 gemcitabine resistant cells. Values are standardized to the control. Values are expressed as mean ± SEM (n = 3). *p< 0.05 vs. control.

Table 9. Docking results with ligands in human Smo and Caspase-3 receptor.

	Glide Gscore (kcal/mol)
	Human Smo					Caspase-3
	5L7I	4QIM	4QIN	4O9R	4N4W	3GJQ
Gemcitabine	−7.491	−7.203	−6.667	−6.571	−7.763	−6.221
Luteolin 7-O-glucoside	−8.387	−6.948	−7.667	−7.728	−8.514	−6.607
Quercetin	−7.678	−8.699	−6.738	−7.126	−8.356	−6.637
Luteolin	−7.639	−8.746	−8.041	−7.008	−8.071	−6.462
Hesperetin	−7.591	−7.935	−7.619	−7.372	−8.341	−7.009
Kaempferol	−7.172	−8.184	−6.826	−7.547	−7.844	−5.889
Trimethoxyflavone	−6.293	−7.98	−6.724	−7.033	−9.091	−4.877
Caffeic acid	−5.628	−5.578	−4.996	−5.022	−6.25	−5.82
Gallic acid	−5.463	−5.746	−5.207	−5.018	−6.363	−7.219
Ferulic acid	−5.037	−4.395	−5.65	−5.39	−5.976	−5.889
syringic acid	−5.013	−4.667	−5.781	−4.905	−5.377	−7.188
Tyrosol	−4.975	−4.872	−3.439	−4.956	−6.081	−3.748
Sinapic acid	−4.747	−4.265	−5.379	−4.809	−6.089	−5.753
Isorhamnetin-3-O-rutinoside	–	–	−7.697	−7.976	–	−9.038

Figure 9. 2D diagrams of ligands interactions with the active sites. (A) Luteolin 7-O-glucoside interactions with the active site of 5L7I. (B and C) Luteolin interactions with the active site of 4QIM and 4QIN, respectively. (D) Isorhamnetin-3-O-rutinoside interactions with the active site of 4O9R. (E) Trimethoxyflavone interactions with the active site of 4N4W. (F) Isorhamnetin-3-O-rutinoside interactions with the active site of the Caspase-3 receptor (3GJQ).

Figure 10. 3D diagrams of ligands interactions with the active sites. (A) Luteolin 7-O-glucoside interactions with the active site of 5L7I. (B and C) Luteolin interactions with the active site of 4QIM and 4QIN, respectively. (D) Isorhamnetin-3-O-rutinoside interactions with the active site of 4O9R. (E) Trimethoxyflavone interactions with the active site of 4N4W. (F) Isorhamnetin-3-O-rutinoside interactions with the active site of the Caspase-3 receptor (3GJQ).